Abstracts
Short talk 4: Towards an evolutionary model of phase separation–driven ribosome biogenesis
Katrina Meyer
Max-Planck-Institute for Molecular Genetics, , Ihnestr. 63, 14195 Berlin [DE], katrina.meyer@molgen.mpg.de
Author(s):
Katrina Meyer, Deana Haxhiraj, Marcel Wittmund, Kiersten Ruff, René Buschow, Nadine Brombacher, Guillermo Pérez-Hernández, Melissa Bothe, Edda Schulz, Denes Hnisz, Rohit Pappu, Rainer Nikolay, Matthew Kraushar
In eukaryotes, 80 ribosomal proteins (RPs) must be efficiently targeted to the nucleolus, where they assemble with four rRNAs to form ribosomes. The nucleolus is a prominent biomolecular condensate, characterized by a multilayered liquid-like architecture. Yet, the molecular features that guide RPs to the nucleolus and their evolution remain poorly understood. To address this, from bacteria to humans,
i) we developed a high-throughput screen for subcellular peptide localization. Our method relies on pooled oligonucleotide synthesis, lentiviral delivery, and stable cell line generation to express tiled peptide libraries. This strategy reliably recapitulates known nucleolar localization signals, aligns with computational predictions, and identifies novel nucleolar-targeting sequences in RPs that had not been previously annotated. The data can be mapped back to ribosomal structures, offering insights into how localization motifs relate to ribosome architecture.
ii) we systematically investigated the condensation behavior of RPs in vitro. We developed a method to purify the full set of RPs en masse circumventing the need for individual expression and purification. Using these preparations, we screened buffer conditions (pH, salt concentration, temperature) to determine how environmental parameters influence condensate formation. Our results indicate subunit-specific properties of RPs as suggested by our initial bioinformatic analyses.